The function and normal regulation of calpain-3, a muscle-specific Ca²⁺-dependent protease, is uncertain, although its absence leads to limb-girdle muscular dystrophy type 2A. This study examined the effect of eccentric exercise on calpain-3 autolytic activation, because such exercise is known to damage sarcomeric structures and to trigger adaptive changes that help prevent such damage on subsequent exercise. Six healthy human subjects performed a 30-min bout of one-legged, eccentric, knee extensor exercise. Torque measurements, vastus lateralis muscle biopsies, and venous blood samples were taken before and up to 7 days following the exercise. Peak isometric muscle torque was depressed immediately and at 3 h postexercise and recovered by 24 h, and serum creatine kinase concentration peaked at 24 h postexercise. The amount of autolyzed calpain-3 was unchanged immediately and 3 h after exercise, but increased markedly (from ∼16% to ∼35% of total) 24 h after the exercise, and returned to preexercise levels within 7 days. In contrast, the eccentric exercise produced little autolytic activation of the ubiquitous Ca²⁺-activated protease, μ-calpain. Eccentric exercise is the first physiological circumstance shown to result in calpain-3 activation in vivo.